Enzymes useful for changing the properties of polyester

ABSTRACT

A method is provided for enzymatically modifying a polyester resin, film, fiber, yarn, fabric or textile to modify the characteristics thereof.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to the field of the modification ofsynthetic polyester used in the production of fibers, yarns, fabrics,films, resins and other objects used for the production of plastics,fabrics, textiles, rugs and other consumer items. More specifically, thepresent invention relates to a new class of enzymes which have theability to modify the surface of polyester resins and fibers andarticles produced therewith.

B. State of the Art

Polyesters are manufactured synthetic compositions comprising any longchain synthetic polymer composed of at least 85% by weight of an esterof a substituted aromatic carboxylic acid, including but not restrictedto substituted terephthalate units and parasubstituted hydroxybenzoateunits. The polyester may take the form of a fiber, yarn, fabric, film,resin or powder. Many chemical derivatives have been developed, forexample, polyethylene terephthalate (PET), polytrimethyleneterephthalate (PTT), polybutylene terephthalate (PBT) and polyethylenenaphthalate (PEN). However, PET is the most common linear polymerproduced and accounts for a majority of the polyester applied inindustry today.

Thermoplastic polyester can be selectively engineered in any of thebasic processing steps of polymerization and fiber formation. Thisflexibility and range of properties allows for a wide range of productsto be made from polyester for markets such as the apparel, homefurnishing, upholstery, film, rigid and flexible container, non-wovenfabric, tire and carpet industries. As a result, polyester has becomethe dominant reinforcement fiber in the United States.

Over the past 30 years cotton has continued slow, steady growth ofvolume consumed and wool has been virtually flat. Polyester, however,has begun to take on increased significance. Moreover, polyester hasreached a higher level of consumer acceptance due to recognition of itsstrength and the increasing quality and variety of fabrics that can bemade using such fibers. Other polyester markets such as fiber-fill andnon-woven articles continue to grow.

In the textile industry, polyester has certain key advantages includinghigh strength, soft hand, stretch resistance, stain resistance, machinewashability, wrinkle resistance and abrasion resistance. However,polyester is not so optimal in terms of its hydrophobicity, pilling,static, dyeability, inactive surface as a medium for adhering, i.e.,softening or wettability enhancing compounds, lack of breathability andundesirable high shine or luster appearance. Moreover, in the 1960's and1970's, polyester textiles suffered from poor consumer perception andwas synonymous with the phrase “cheaply made” and derided for thehorrendous colors with which polyester was associated. This latterproblem is due in large part to the unavailability of a large selectionof dyes which are compatible with polyester. To combat this perception,the industry has made strong efforts to improve the characteristics ofpolyester.

One attribute that the industry has sought to achieve with treatments ofpolyester is to provide depilling and pilling prevention to polyestergarments. The industry has also sought to improve the hand and feel ofpolyester, for example, by decreasing the weight of polyester fabrics.Another problem area involves the characteristic that polyester is veryresistant to uptake of polar or charged compositions, i.e., fabricsofteners, finishes and dyes. Another problem with polyester relates tothe difficulty of removing oily and/or hydrophobic stains. These stainsoften adhere strongly to the fabric or fiber and cause a permanentstain.

GB 2296011 A discloses enzymes naturally produced by a fungus of thespecies Fusarium solanii var. minus T.92.637/1, including a cutinase ofisoelectric point 7.2 and mol. wt. 22 kDa. which are useful in detergentcompositions for removing fatty acid-based dirt and stains.

U.S. Pat. No. 5,512,203 discloses cleaning compositions comprising acutinase enzyme and a cutinase compatible surfactant. The microbialcutinase is from Pseudomonas mendocina and is used in an improved methodfor enzymatically cleaning a material having a cutin or cutin-likestain.

PCT Publication No. WO 97/43014 (Bayer AG) describes the enzymaticdegradation of polyesteramide by treatment with an aqueous solutioncomprising an esterase, lipase or protease.

JP 5344897 A (Amano Pharmaceutical KK) describes a commercial lipasecomposition which is dissolved in solution with an aliphatic polyesterwith the result that the fiber texture is improved without losingstrength. Polymers of aliphatic polyethylene are also disclosed whichcan be degraded by lipase from Pseudomonas spp.

PCT Publication No. 97/33001 (Genencor International, Inc.) discloses amethod for improving the wettability and absorbency of a polyesterfabric by treating with a lipase.

PCT Publication No. WO 99/01604 (Novo Nordisk) describes a method fordepilling a polyester fiber or fabric and for color clarification indetergents of such fabrics by reacting with an enzyme which hashydrolytic activity on either ethyleneglycol dibenzyl ester (BEB) and/orterephthalic acid diethyl ester (ETE) subunit components.

As can be seen from the above, many advances have been made in thetreatment of polyester to improve its properties. In addition, there hasbeen some work in the area of using enzymes to achieve such results.However, this work has focused on the ability of enzymes to degrademono- and di-ester subunits. Applicants have surprisingly discoveredthat a great number of enzymes which have mono- and/or di-esterhydrolysis activity do not have polyester modification properties. Incontrast, Applicants discovered that a true polyesterase enzyme whichhas the ability to modify polyester cannot be selected merely on theirability to hydrolyze mono- and/or di-esters and must be selected usingdifferent criteria.

Thus, despite the considerable work done in the field, the industryremains in need of additional methods of producing modified polyesterswith improved characteristics. For example, with respect to textiles,such improvement may relate to pilling prevention, de-pilling duringmanufacture, increase in desirable hand and feel and appearance,improved static resistance, increased ability to uptake hydrophilicsubstances, improved more natural luster and improved oily stainresistance.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide for an enzymecomposition which has excellent ability to modify polyester fiber andresin properties.

It is a further object of the present invention to provide for a methodof enzymatically modifying the properties of an article comprisingpolyester fiber or resin.

It is a further object of the invention to provide for a method oftreating the surface of a polyester fiber article such that the articlehas modified characteristics with respect to pilling prevention anddepilling characteristics.

It is a further object of the invention to provide for a method ofproducing polyesters having unique functionalities such as having lessstatic cling and a more natural looking appearance, e.g., being lessshiny or having a more natural “luster”.

It is yet a further object of the invention to provide for a polyesterfiber or resin composition that, upon being woven into a textilearticle, has modified weight, hand and/or feel.

It is yet a further object of the invention to provide for methods ofachieving such effects on unsoiled fabrics and/or during manufacture.

According to the present invention, a method is provided for treating aclean, unsoiled polyester comprising contacting said polyester textilewith an enzyme solution having polyesterase activity for a time andunder conditions such that the properties of the polyester are modified.Preferably, the polyester is a fiber, yarn, fabric or finished textileproduct comprising such fiber, yarn or fabric. Further preferably, theproperties that are modified comprise those such as improved hand, feeland/or weight of a textile made from such fiber, yarn or article.Preferably, the textile properties of the fiber, yarn or fabric aremodified.

In another embodiment of the invention, a method is provided fortreating a polyester fiber, yarn or fabric, prior to its incorporationinto a textile product or the application of a textile finish with anenzyme having polyesterase activity for a time and under conditions suchthat the properties of the polyester are modified. Accordingly, in theembodiment wherein textile components are treated separately, thetreated polyester components (i.e., fibers, yarns, fabrics), may beincorporated into a textile product through standard methods forproducing polyester textiles, thus conferring the modifications to thefinished textile product. Preferably, the textile properties of thefiber, yarn or fabric are modified.

In yet another method embodiment of the invention, a method is providedfor treating a polyester resin or film with an enzyme havingpolyesterase activity for a time and under conditions such that theproperties of the polyester are modified. The treated polyester may be afinished resin or film product or may be incorporated into a productthrough, for example, mechanical construction, thus conferring themodifications to the finished textile product.

In yet another method embodiment of the invention, a polyester wasteproduct is treated with the polyesterase enzyme of the invention todegrade the polyester waste product to easily disposed of or recycledcompounds. This embodiment is particularly useful in the degradation ofpolyester based plastics which are becoming increasingly problematic inwaste disposal and dumping. An alternative of this embodiment is thatthe present invention may be used to increase the amount of microbiallydigestible material in a waste product so as to facilitate completedegradation or composting of such waste.

In yet another method embodiment of the invention, a polyester isproduced from monomer units by reversing the equilibrium of the reactionusing the polyesterase.

In a composition embodiment of the invention, a polyester article isprovided according to the method of the invention. Preferably, thepolyester article has improved weight, hand, feel, depilling or pillingprevention properties.

In a further embodiment of the invention, a method is provided for thepolymerization of polyester fibers using an enzymatic catalyst.

In yet a further embodiment of the invention, a method is provided forremoving a sizing material from a textile which sizing materialcomprises a polyester composition.

In a method embodiment of the invention, an assay is provided for theisolation and/or determination of a polyesterase enzyme. In acomposition embodiment of the invention, a kit is provided for carryingout the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of polyesterase treatments on thedyeability of Dacron 54.

FIG. 2 illustrates the effect of polyesterase treatments on thedyeability of Dacron 64.

FIG. 3 illustrates comparative quantitative hydrolysis product frompolyesterase enzyme treatment of Dacron 54.

FIG. 4 illustrates weight loss of a polyester after treatment with apolyesterase enzyme.

FIG. 5 illustrates a scanning electron micrograph of polyester fiberincubated w/100 mM Tris Buffer (pH 8.6/40° C.). 1000×.

FIG. 6 illustrates a scanning electron micrograph of polyester fiberincubated with Tris Buffer+Cutinase (pH 8.6/40° C.). 1000×.

FIG. 7 illustrates a scanning electron micrograph of polyester fiberincubated with buffer and glycerol (50/50 w/w). 500×.

FIG. 8 illustrates a scanning electron micrograph of polyester fibertreated w/buffer and glycerol and cutinase. 500×.

FIG. 9 illustrates the effect of polyesterase treatments on thedyeability of Corterra™ fabric.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a method is provided for treating aclean, unsoiled polyester comprising contacting said polyester textilewith an enzyme solution having polyesterase activity for a time andunder conditions such that the properties of the polyester are modified.Preferably, the polyester is a fiber, yarn, fabric or finished textileproduct comprising such fiber, yarn or fabric. Further preferably, theproperties that are modified comprise those such as improved hand, feeland/or weight of a textile made from such fiber, yarn or article. Thepurpose of this embodiment of the present invention is not to providefor a method of laundering stains from polyester fabrics, but instead,to provide for a mechanism to modify the textile characteristics of apolyester comprising textile. Thus, in this embodiment of the invention,it is often advantageous to apply the polyesterase to textile productswhich are unsoiled, i.e., do not comprise stains which are typicallysubjected to commercial laundry detergents.

In another embodiment of the invention, a method is provided fortreating a polyester fiber, yarn or fabric, prior to its incorporationinto a textile product or the application of a textile finish with anenzyme having polyesterase activity for a time and under conditions suchthat the properties of the polyester are modified. Accordingly, in theembodiment wherein textile components are treated separately, thetreated polyester components (i.e., fibers, yarns, fabrics), may beincorporated into a textile product through standard methods forproducing polyester textiles, e.g., processes such as weaving, sewingand cutting and stitching, thus conferring the modifications to thefinished textile product.

In yet another method embodiment of the invention, a method is providedfor treating a polyester resin or film with an enzyme havingpolyesterase activity for a time and under conditions such that theproperties of the polyester are modified. The treated polyester may be afinished resin or film product or may be incorporated into a productthrough, for example, mechanical construction, thus conferring themodifications to the finished textile product.

In yet another method embodiment of the invention, a polyester wasteproduct is treated with the polyesterase enzyme of the invention todegrade the polyester waste product to easily disposed of or recycledcompounds. This embodiment is particularly useful in the degradation ofpolyester based plastics which are becoming increasingly problematic inwaste disposal and dumping. An alternative of this embodiment is thatthe present invention may be used to increase the amount of microbiallydigestible material in a waste product so as to facilitate completedegradation or composting of such waste.

In the method according to the invention, the polyesterase solution asprovided herein is contacted with the polyester fiber, yarn, fabric ortextile which comprises such fiber, yarn or fabric under conditionssuitable for the enzyme to exhibit polyester modification. The presentinvention is preferably directed to the use of the polyesterase in themanufacture of the textile product, and not necessarily in combinationwith a detergent for the purpose of removing stains which occur duringwear. Thus, in this embodiment, the application of the polyesterase tothe polyester article occurs prior to spinning of the fiber into a yarn,prior to the incorporation of the yarn into a fabric and/or prior to theconstruction of the textile product which comprises the polyester.However, it is within the present invention as well, and also apreferred embodiment hereon, to treat the completed textile product withthe polyesterase identified herein.

In another embodiment of the invention, the polyesterase reaction is runso that the equilibrium of the catalytic reaction is shifted towards theproduction of polyester from monomer subunits. Such equilibrium shiftscan be accomplished by one of ordinary skill in the art using routineenzymological and chemical methods including optimization of organicsolvents and supercritical fluids.

“Polyester” as used herein means a linear polymeric molecule containingin-chain ester groups and which are derived from the condensation of adiacid with a diol or from the polymerization of hydroxy acids. Thepresent invention applies to both aliphatic and aromatic polyesters.However, particularly preferred are aromatic polyester articles whichare used to produce fiber and resin and that comprise a syntheticallyproduced long chain polymer comprising at least 85%, preferably at least90% and most preferably at least 95%, by weight of an ester of asubstituted aromatic carboxylic acid, such as substituted terephthalicacid or parasubstituted hydroxybenzoate. Other useful polyester articlesinclude those made of bulk polymer, yarns, fabrics, films, resins andpowders. The principal polyesters in industrial usage includepolyethylene terephthalate (PET), tetramethylene terephthalate (PTMT),polybutylene terphthalate (PBT), polytrimethylene terephthalate (PTT)and polyethylene naphthalate (PEN), polycyclohexanedimethyleneterephthalate (CHDMT), poly(ethylene-4-oxybenzoate) A-Tell,polyglycolide, PHBA and 2GN. Polyester as used herein may take the formof fiber, yarn, fabric, textile article, or any other compositionwherein polyester fibers, yarns or fabrics are employed.

“Polyesterase” means an enzyme that has significant capability tocatalyze the hydrolysis and/or surface modification of PET.Specifically, Applicants have discovered that enzymes which havehydrolytic activity against PET under the conditions provided in the UVand MB assays provided in Example 1(a) and 1(b) (referred to herein asthe “UV Assay” and the “MB Assay” respectively) are useful in thetreatment of polyester resins, films, fibers, yarns and fabrics tomodify the properties thereof. Accordingly, the assays provided inExample 1(a) and 1(b) may be used to isolate polyesterase enzymes and/ordetermine the polyesterase activity of an enzyme.

Applicants have surprisingly found that enzymes according to the presentinvention represent a subclass of enzymes which have significantactivity against polyester and are capable of producing improved surfacemodification effects. By contrast, enzymes defined by prior art assaysappear to be more general and to have a greater instance of falsepositive results. Assays designed to measure hydrolysis of mono- anddi-ester units, such as the assays measuring ETE and BEB hydrolysisdescribed in WO 99/01604, are useful in identifying a large number ofenzymes, some of which may fortuitously have useful polyesteraseactivity. However, these assays are based on hydrolysis of mono- anddi-ester molecules. As a consequence, these results are often notpredictive of the likelihood that a specific enzyme will successfullymodify the surface of long chain polyesters. Example 1 (d) shows thatassays designed on small molecule hydrolysis will broadly includeenzymes which are useful against the mono- and di-ester molecules whilenot predicting with accuracy whether such enzymes have activity againstlarge repeating polymer fibers such as long chain polyesters.

Thus, the polyesterase enzymes of the present invention will produce apositive result according to one or both of the polyesterase assaysdescribed herein. The activity of the enzymes of the invention insolution will produce an absorbance of at least 10% above the controlblank, preferably 50% and most preferably 100% greater than the controlblank. In a most preferred embodiment, the polyesterase enzymes of theinvention will produce a positive result in both assays which is atleast double the increase in absorbance reading of the blank sample.

Suitable polyesterases may be isolated from animal, plant, fungal andbacterial sources. With respect to the use of polyesterases derived fromplants, polyesterases may exist in the pollen of many plants.Polyesterases may also be derived a fungus, such as, Absidia spp.;Acremonium spp.; Agaricus spp.; Anaeromyces spp.; Aspergillus spp.,including A. auculeatus, A. awamori, A. flavus, A. foetidus, A.fumaricus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreusand A. versicolor; Aeurobasidium spp.; Cephalosporum spp.; Chaetomiumspp.; Cladosporium spp.; Coprinus spp.; Dactyllum spp.; Fusarium spp.,including F. conglomerans, F. decemcellulare, F. javanicum, F. lini, F.oxysporum, F. roseum and F. solani; Gliocladium spp.; Helminthosporumspp., including sativum; Humicola spp., including H. insolens and H.lanuginosa; Mucor spp.; Neurospora spp., including N. crassa and N.sitophila; Neocallimastix spp.; Orpinomyces spp.; Penicillium spp;Phanerochaete spp.; Phlebia spp.; Piromyces spp.; Pseudomonas spp.;Rhizopus spp.; Schizophyllum spp.; Trametes spp.; Trichoderma spp.,including T. reesei, T. reesei (longibrachiatum) and T. viride; andUlocladium spp., including U. consortiale; Zygorhynchus spp. Similarly,it is envisioned that a polyesterase may be found in bacteria such asBacillus spp.; Cellulimonas spp.; Clostridium spp.; Myceliophthora spp.;Pseudomonas spp., including P. mendocina and P. putida; Thermomonosporaspp.; Thermomyces spp., including T. lanuginosa; Streptomyces spp.,including S. olivochromogenes and S. scabies; and in fiber degradingruminal bacteria such as Fibrobacter succinogenes; and in yeastincluding Candida spp., including C. Antarctica, C. rugosa, torresii; C.parapsilosis; C. sake; C. zeylanoides; Pichia minuta; Rhodotorulaglutinis; R. mucilaginosa; and Sporobolomyces holsaticus.

“Textile” means any fabric or yarn or product which incorporates afabric or yarn. Examples of textiles which may be treated with thepresent invention include clothing, footwear, upholstery, draperies,carpets, outdoor gear, ropes and rope based products. As used in thepresent invention, textile includes non-woven fabrics used in, forexample, the medical industry.

“Biological material” means any composition which is derived frombiological origin, including, but not limited to, cells, vectors, DNA,protein, cell membranes, cellular components, RNA or any mixturecomprising such materials.

“Textile properties” means the properties of a textile comprising apolyester fiber, yarn or fabric that are critical to the appearance,feel or comfort of the article. As used herein, textile propertiesincludes depilling, antipilling, improvement of hand, improvement offeel, improvement of appearance such as luster and drape, improving thewettability or absorbency, decreasing static cling, decreasing oily soilattraction and improving soil release properties or otherwise creating aunique appearance by physical modification of the polyester in a mannerso as to improve the textile in manner that provides unique features tothe textile.

“Treatment” means with respect to treatment with polyesterase comprisesthe process of applying the polyesterase to the polyester article suchthat the enzyme is capable of reacting with the surface of the polyesterarticle to such an extent that the properties of the article aresignificantly improved. Generally, this means that the polyesterase ismixed with the polyester article in an environment that facilitates theenzymatic action of the polyesterase. Such conditions may be readilydetermined through routine testing by the skilled enzymologist. In thecontext of fibers, yarns or fabrics used in the production of a textile,in a preferred embodiment the textile properties are modified. In thecontext of a resin or a film, the surface characteristics of thepolyester film or resin are modified to, e.g., modify the hydrophilicityof the surface of its ability to adhere charged coatings or othersubstances to the surface.

“Textile finish” means sizing agents, lubricants, defoaming agents,anti-static agents and other compositions added to polyester fibers,yarns or fabrics during the manufacture of consumer or industrialproducts.

Treating according to the instant invention may comprise preparing anaqueous solution (or organic solvent or mixtures of organic compounds)that contains an effective amount of a polyesterase or a combination ofpolyesterases together with other optional ingredients including, forexample, a buffer or a surfactant. An effective amount of a polyesteraseenzyme composition is a concentration of polyesterase enzyme sufficientfor its intended purpose. Thus, for example, an “effective amount” ofpolyesterase in a composition intended to produce depilling over aseries of washes according to the present invention is that amount whichwill provide the desired effect, e.g., to improve the textile propertiesof the polyester containing textile article in comparison with a similarmethod not using polyesterase or to improve the surface properties of afilm or resin. The amount of polyesterase employed is also dependent onthe equipment employed, the process parameters employed, e.g., thetemperature of the polyesterase treatment solution, the exposure time tothe polyesterase solution, and the polyesterase activity (e.g., aparticular solution will require a lower concentration of polyesterasewhere a more active polyesterase composition is used as compared to aless active polyesterase composition). The exact concentration ofpolyesterase in the treatment solution can be readily determined by theskilled artisan based on the above factors as well as the desiredresult. However, it has been observed by the inventors herein that thebenefit disclosed herein requires a relatively rigorous polyesterasetreatment. Thus, the benefits described herein are not likely to beshown with modest concentrations of polyesterase and relatively short(less than one hour) treatment times with presently characterizedenzymes. Nonetheless, it is possible and preferable that an engineeredpolyesterase or a polyesterase with exceptionally high activity onpolyester under given conditions could be obtained which would requireless than 1 hour of treatment to reach the desired benefit levels andthus fall within the scope of the present invention. Similarly,employing large amounts of polyesterase for relatively short periods oftime may also result in achievement of the benefits described herein.

In one treating embodiment, a buffer may be employed in the treatingcomposition such that the concentration of buffer is sufficient tomaintain the pH of the solution within the range wherein the employedpolyesterase exhibits the desired activity. The pH at which thepolyesterase exhibits activity depends on the nature of the polyesteraseemployed. The exact concentration of buffer employed will depend onseveral factors which the skilled artisan can readily take into account.For example, in a preferred embodiment, the buffer as well as the bufferconcentration are selected so as to maintain the pH of the finalpolyesterase solution within the pH range required for optimalpolyesterase activity. The determination of the optimal pH range of thepolyesterase of the invention can be ascertained according to well knowntechniques. Suitable buffers at pH within the activity range of thepolyesterase are also well known to those skilled in the art in thefield.

In addition to polyesterase and a buffer, the treating composition maycontain a surfactant, i.e., a cationic, nonionic or anionic surfactant.Suitable surfactants include any surfactant compatible with thepolyesterase being utilized and the fabric including, for example,anionic, non-ionic and ampholytic surfactants. Suitable anionicsurfactants include, but are not limited to, linear or branchedalkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear orbranched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates;olefinsulfonates; alkanesulfonates and the like. Suitable counter ionsfor anionic surfactants include, but are not limited to, alkali metalions such as sodium and potassium; alkaline earth metal ions such ascalcium and magnesium; ammonium ion; and alkanolamines having 1 to 3alkanol groups of carbon number 2 or 3. Ampholytic surfactants include,e.g., quaternary ammonium salt sulfonates, and betaine-type ampholyticsurfactants. Such ampholytic surfactants have both the positive andnegative charged groups in the same molecule. Nonionic surfactantsgenerally comprise polyoxyalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, and fatty acid glycerinemonoesters. Mixtures of surfactants can also be employed in mannersknown to those skilled in the art.

In a particularly preferred embodiment of the invention, it is desirableto add glycerol, ethylene glycol or polypropylene glycol to the treatingcomposition. Applicants have discovered that the addition of glycerol,ethylene glycol, or polypropylene glycol contributes to enhancedactivity of the polyesterase on polyester. Applicants have determinedthat defoaming agents and/or lubricants such as Mazu® have a desirableeffect on the activity of the polyesterase.

In some embodiments, it may be desirable to adjust the parametersdiscussed above for the purpose of controlling the enzymaticdegradation. For example, the pH can be adjusted at certain time pointsto extinguish the activity of the polyesterase and prevent undesirableexcessive degradation. Alternatively, other art recognized methods ofextinguishing enzyme activity may be implemented, e.g., proteasetreatment and/or heat treatment.

As can be seen above, the present invention is useful in the preparationof laundry detergents. For example, it may be desirable to encourage theuptake of a cationic laundry adjuvant, i.e., a fabric softener or othersuch compounds which improve the feel, appearance or comfort oflaundered fabrics. In this case, the present invention will provide formethods to modify the polyester during the wash cycle so as to encouragethe uptake of the advantageous adjuvant.

EXAMPLES Example 1

This Example provides for two assays which identify polyesteraseactivity in a potential enzyme candidate. Preferably, the enzyme willshow polyester hydrolysis activity in both assays.

(A) Assay for Enzymatic Hydrolysis of Long Chain Polyester PolymerFibers Based on Ultraviolet Light Absorbance (UV Assay)

This assay monitors the release of terephthalate and its estersresulting from the enzymatic hydrolysis of polyester and measures thehydrolysis product by subjecting the sample to the UV spectrum andmeasuring absorbance.

Materials:

-   -   Enzyme reaction buffer: 100 mM Tris, pH 8, optionally containing        0.1% Brij®-35

Procedure:

-   -   1. The polyester is washed with hot water and air dried.        Applicants recommend and exemplify herein the use of such easily        obtained standardized polyesters as Dacron® 54 woven polyester        (from Testfabrics) (used in the description below). However, it        will often be preferable to use the specific polyester substrate        for which modification is desired, e.g., fabric, powder, resin        or film, thereby ensuring that the enzyme selected will have        optimal activity on that specific substrate. In such case, it is        merely necessary to substitute the desired polyester substrate        for the below described Dacron.    -   2. ⅝-inch circular swatches are cut from the Dacron® 54.    -   3. The swatches are incubated in reaction buffer in sealed        12-well microtiter plates with orbital shaking at 250 rpm. A        typical reaction is 1 mL in volume, with 10 μg enzyme. Three        samples should be run: (1) substrate+buffer, (2)        enzyme+buffer, (3) enzyme+substrate+buffer.    -   4. The reaction is allowed to proceed for 18 hours at 40° C.    -   5. Terephthalate and its esters have characteristic strong        absorbance peaks around 240-244 nm (ε_(M)˜10,000). Therefore, if        these species are released to the liquid phase of the reaction        by enzymatic hydrolysis, the absorbance of liquid phase of the        reaction will be increased at these wavelengths.    -   6. To determine if hydrolysis has occurred, one determines the        absorbance of the liquid phase of the enzyme+substrate+buffer        reaction at around 240-250 nm. The appropriate blanks        (substrate+buffer, and enzyme+buffer) must be subtracted. These        measurements can be carried out in a quartz cuvette in a        spectrophotometer or a UV-transparent microtiter plate in a        microplate reader capable of the required wavelengths.    -   7. To confirm that the absorbance readings higher than the        blanks are actually due to terephthalate compounds, an        absorbance spectrum of the reaction mixture should be scanned        from 220-300 nm. Only a peak around 240-244 nm should be        considered as actual reaction product.    -   8. Terephthalic acid and diethyl terephthalate are commercially        available. Their absorbance spectra should serve as standards.        (B) Assay for Enzymatic Hydrolysis of Long Chain Polyester        Polymer Fibers Based on Binding of Methylene Blue (MB Assay)

This assay utilizes the binding of methylene blue, a cationic dye, tothe free carboxylate groups generated by hydrolysis of polyester.

Materials:

-   -   Enzyme reaction buffer: 100 mM Tris, pH 8, containing 0.1%        Triton® X-100    -   Wash buffer: 100 mM MES, pH 6.0    -   Dye solution: 0.1 mg/mL methylene blue in 1 mM MES, pH 6.0    -   Dye elution buffer: 0.5 M NaCl in 10 mM MES, pH 6.0    -   Dacron 54 woven polyester from Testfabrics.

Procedure:

-   -   1. The polyester is washed with hot water and air dried.        Applicants recommend the use of such easily obtained        standardized polyesters as Dacron® 54 woven polyester (from        Testfabrics) (used in the description below). However, it will        often be preferable to use the specific polyester substrate for        which modification is desired, e.g., fabric, powder, resin or        film, thereby ensuring that the enzyme selected will have        optimal activity on that specific substrate.    -   2. ⅝-in. circular swatches are cut from the Dacron®.    -   3. The swatches are incubated in reaction buffer in sealed        12-well microtiter plates with orbital shaking at 250 rpm. A        typical reaction is 1 mL in volume, with 10 μg enzyme. Blanks        (samples with no enzyme) should be run as well.    -   4. The reaction is allowed to proceed for 18 hours at 40° C.    -   5. The reaction solution is removed by suction, and the swatches        are subsequently washed with: (1) 1 ml incubation buffer, to        deplete residual enzyme; (2) 1 ml water, to deplete the        incubation buffer; (3) 1 ml 100 mM MES buffer, to equilibrate        the swatches to pH 6; and (4) 1 ml water again, deplete the MES        buffer.    -   6. 1 mL of dye solution is added to each well, and the plate is        shaken at 250 rpm for 20 min at 40° C. In this case, methylene        blue is used. However, other cationic dyes or “reporter”        reagents can be used as well. Hydrolysis by 100 mM NaOH can be        used as a positive control.    -   7. The excess dye (methylene blue) is removed by suction, and        the wells are washed 3 times with 1 ml water.    -   8. 1 mL dye elution buffer is added to each well, and the plate        is shaken at 250 rpm for 30 min at 40° C.    -   9. 300 μL of the dye eluate is transferred from each well to a        96-well plate, and the absorbance peak at 650 nm is determined.

In either of the above assays described in Examples 1(a) and 1(b), theabsorbance reading should show significant hydrolytic product which isnot attributable to experimental error or non-hydrolytic effects. One ofskill in the art is well aware of these effects and how to guard againstthem in interpreting results.

(C) Assay for Enzymatic Hydrolysis of Diethyl Terephthalate (DET)

This spectrophotometric assay monitors the change in the UV spectrum ofDET which accompanies its hydrolysis.

DET has a characteristic absorbance peak around 244 nm ε_(M)˜10,000).The ester hydrolysis products have a lower absorbance, and the peak isshifted to 240 nm. Consequently, the hydrolysis of DET can be monitoredby measuring the decrease in absorbance at 250 nm.

Reagents:

-   -   Enzyme reaction buffer: 10 mM Tris, pH 8    -   DET stock solution: 100 mM in DMSO

Procedure:

-   -   1. Dilute DET 1000-fold into reaction buffer to yield a 100 μM        solution. Place in a cuvette or UV transparent microtiter plate.    -   2. Set the spectrophotometer wavelength at 250 nm.    -   3. Add enzyme, and monitor the change in absorbance. In a        separate sample of the same volume of buffer without enzyme,        determine the absorbance change resulting from background        hydrolysis.    -   4. Reaction rate is calculated from the linear portion of the        reaction progress curve and reported as −mAU/min and the        reaction rate of the buffer blank is subtracted.        (D) Comparison of Results of PET and DET Assays

Enzymes having esterase and/or lipase activity were obtained fromnumerous sources and tested according to the assays described inExamples 1(a), 1(b) and 1(c). The relative results are tabulated inTable I with the hydrolysis product absorbance of P. mendocina cutinasebeing calculated as 1.0 under the conditions used. TABLE I Enzyme OriginClass DET PET (UV) PET (MB) Blank/Control <0.3 <0.1 <0.4 PseudomonasCutinase 1.0 1.0 1.0 mendocina Pseudomonas sp Lipase 1.2 0.2 <0.4Pseudomonas Lipase <0.3 0.1 <0.4 fluorescens Aspergillus niger Esterase0.8 <0.1 <0.4 Candida antarctica Lipase A <0.3 <0.1 <0.4 Candidaantarctica Lipase B 2.3 <0.1 <0.4 Candida lipolytica Lipase 0.1 <0.1<0.4 Candida rugosa Lipase 0.8 <0.1 0.5 Candida rugosa Lipase, purif.2.2 <0.1 <0.4 Humicola Lipase 0.3 <0.1 <0.4 lanuginosa Rhizopus delmarLipase 0.7 <0.1 <0.4 Rhizopus Lipase 0.7 <0.1 <0.4 javanicus Rhizopusniveus Lipase 0.8 <0.1 <0.4 Mucor meihei Lipase <0.3 <0.1 <0.4 WheatGerm Lipase 0.6 <0.1 <0.4 Lipolase ™¹ Lipase 1.2 <0.1 <0.4 Lipomax ™²Lipase 2.7 <0.1 0.7 Pig Pancreas Lipase 1.0 <0.1 <0.4 Pig Liver³Esterase I 3.1 <0.1 <0.4 Pig Liver Esterase II 2.0 <0.1 <0.4 E001⁴Esterase 2.3 <0.1 <0.4 E002 Esterase 3.3 <0.1 <0.4 E003 Esterase 5.0<0.1 <0.4 E004 Esterase 1.2 <0.1 <0.4 E005 Esterase 1.3 <0.1 <0.4 E006Esterase 2.7 <0.1 <0.4 E007 Esterase 2.4 <0.1 <0.4 E008 Esterase 2.0<0.1 <0.4 E009 Esterase 1.5 <0.1 <0.4 E010 Esterase 2.6 <0.1 <0.4 E011Esterase 4.0 0.1 <0.4 E012 Esterase 1.1 <0.1 <0.4 E013 Esterase 2.4 <0.1<0.4 E014 Esterase 5.2 <0.1 <0.4 E015 Esterase 3.6 <0.1 <0.4 E016Esterase 2.0 <0.1 <0.4 E017b Esterase 3.7 <0.1 <0.4 E018b Esterase 0.6<0.1 <0.4 E019 Esterase 0.9 <0.1 <0.4 E020 Esterase 2.0 <0.1 <0.4ESL-001-01⁵ Esterase 0.7 <0.1 <0.4 ESL 001-02 Esterase 4.6 <0.1 <0.4ESL-001-03 Esterase 0.6 <0.1 <0.4 ESL 001-04 Esterase 1.3 <0.1 <0.4 ESL001-05 Esterase 0.9 <0.1 <0.4 ESL 001-06 Esterase 0.4 <0.1 <0.4 ESL001-07 Esterase 0.9 <0.1 <0.4 Chiro-CLEC-CR⁶ EC 3.1.1.3 0.5 <0.1 <0.4Chiro-CLEC-BL EC 3.4.21.14 <0.3 <0.1 <0.4 Chiro-CLEC-PC EC 3.1.1.3 0.80.1 <0.4 Chiro-CLEC-EC EC 3.5.1.11 0.7 <0.1 <0.4¹(commercial product obtained from Novo Nordisk)²(commercial product obtained from Genencor International, Inc.)³(Pig Liver Esterase I and II obtained from Boehringer MannheimChiraZyme ™ Lipases & Esterases Screening Set (Germany))⁴(All E series esterases listed were obtained from the ThermoCat ™ R&Dproduct line from Thermogen (Chicago, IL))⁵(All “ESL” series esterases were obtained from Diversa Esterase/LipaseCloneZyme ™ Library)⁶(All ChiroCLEC ™ enzymes obtained from Altus Corp ChiroScreen ™ EnzymeSet (Cambridge, Massachusetts))

As can be seen from the above, nearly all of the enzymes tested haveactivity in the DET assay (di-esterase activity). However, only one ofthe tested enzymes has significant activity in both of the PET assays.From this evidence, it is apparent that, while there is cross over interms of enzymes which have activity in the DET assay and also have PEThydrolytic activity, there are a great number of enzymes which do haveDET hydrolytic activity but do not have polyesterase activity. As shownin Examples 2 and 3, the enzyme with PET activity provides significantenzymatic conversion of the polyester fibers. From this data, Applicantsdetermined that the identity of an enzyme having polyesterase activitycannot be predicted from whether that enzyme has mono- or di-esteraseactivity.

Example 2

Enzymatic Surface Modification of Polyester Fibers with Polyesterase toModify the Functional Surface Properties of the Polyester

-   -   Equipment: Launder-Ometer    -   Treatment pH: pH 8.6 (50 mM Tris Buffer)    -   Treatment temperature: 40° C.    -   Treatment time: 24 hours    -   Enzyme: Cutinase from Pseudomonas mendocina @ 40 ppm    -   Control: Inactivated cutinase (Pseudomonas mendocina) @ 40 ppm    -   Substrates: 100% Polyester        -   Dacron® 54 ( style number 777 from TestFabrics)        -   Dacron® 64 (style number 763 from TestFabrics)

To ensure that all observed effects were due solely to the modificationof the polyester surface, and not from adhered protein effects, theswatches were treated with protease. After the polyesterase treatments,⅝ inch disks were cut from the treated swatches. Then the disks wereincubated with 5 ppm subtilisin and 0.1% non-ionic surfactant (TritonX-100) to remove proteins bound onto polyester. The levels of boundproteins were examined using coomassie blue staining to ensure thatminimal protein remained bound to the fabric.

After enzyme treatment followed by protease/surfactant treatments, thedisks were dyed in 12 well microtiter plate under the followingconditions:

-   -   Liquor ratio: 40 to 1    -   Dye concentration: 0.4% owf    -   Temperature: 40° C.    -   pH: 6 (1 mM MES buffer at pH 6.0)    -   Time: 20 minutes    -   Agitation of shaker: 200 rpm

The disks were rinsed three times with DI water after dyeing, air dried,and then measured for CIE L*a*b* values using a reflectometer. The totalcolor difference was calculated using the following formula:Delta E=Square Root (ΔL* ² +Δa* ² +Δb* ²)

ΔL=Difference in CIE L* values before and after dyeing

Δa=Difference in CIE a* values before and after dyeing

Δb=Difference in CIE b* values before and after dyeing

(These terms are defined in, for example, Duff & Sinclair, Giles'sLaboratory Course in Dyeing, 4th Edition, Society of Dyers andColourists). TABLE 1 Total Color Difference after Dyeing with DifferentBasic Dyes Total Color Difference (ΔE) Dacron 54 Dacron 64 Basic DyesCon- Cuti- Con- Cuti- Dye classes trol nase trol nase Methylene Blue8.37 14.66 20.28 25.10 C.I. Basic Yellow 28 (Monazo) 10.72 20.05 26.3232.09 C.I. Basic Yellow 29 (Methine) 9.99 20.35 28.17 34.92 C.I. BasicOrange 42 (Azo-methine-azo) 20.75 27.15 33.04 39.81 C.I. Basic Orange 48(Azo) 10.92 21.41 20.30 26.15 C.I. Basic Blue 45 (Anthraquinone) 10.1810.27 17.06 21.21 C.I. Basic Blue 77 (Triarylmethane) 20.53 27.59 28.8140.89

The results are compiled graphically in FIGS. 1 and 2. As can be seen,polyesterase significantly effects the ability of the polyester fabricsto take up and adhere a range of cationic dyes.

Example 3

Enzyme Catalyzed Polyester Treatment

(A) Enzymatic Polyester De-Pilling

To demonstrate de-pilling of polyester by a polyesterase, the followingexperiments were done using 24 hour pre-pilled Dacron® 54* swatches at40° C. Each Launder-Ometer cycle was taken for 24 hours, and three(5″×7″) swatches were added per experiments.* Dacron® 54 is 100% disperse dyeable polyester manufacture by Dupont.

-   -   Exp. 1) 50 mM Tris Buffer (pH 8.6)+0.01% sodium azide    -   Exp. 2) 50 mM Tris Buffer (pH 8.6)+0.01% sodium        azide+polyesterase (cutinase from P. mendocina)    -   Exp. 3) 20 mM NaOH

After each cycle, the swatches were rinsed with DI water, and thentransferred into autoclaved Launder-Ometer canisters with freshsolution.

NaOH treated swatches (Exp. 1) exhibited clear de-pilling effect after5th cycle, and the experiment was stopped. After each cycle, absorbanceof treated liquor at 250 nM were measured to quantify PET hydrolysis.

Clear differences in de-pilling were observed between buffer control(Exp.1) vs. polyesterase treated (Exp. 2) swatches. The experiments werestopped after 14th cycle.

These results show that the polyesterase treatment produced a markedimprovement in depilling as compared to the treatment with control. Inaddition, as shown in FIG. 3, the improvement in depilling correspondedto the accumulation of PET hydrolysis products in the liquor, confirmingthat the improvement in pilling characteristics was the result of thehydrolytic action of the enzyme on the polyester.

(B) Enzyme Mediated Polyester Fabric Weight Loss

After the Launder-Ometer experiments described above, treated swatches(three per experiment) were weighed individually and the average weightloss was calculated. The results are shown in FIG. 4.

Both polyesterase treated and NaOH treated swatches showed significantweight loss compared to the buffer control and pre-pilled swatches(p<0.05).

(C) Electron Microscopy Showing Polyesterase Caused Surface Modification

PET fibers incubated with 1) buffer and 2) buffer+P. mendocina cutinasefor a month at 40° C. were photographed using scanning electronmicroscopy. The results are provided in FIGS. 5-8.

(D) Hydrolysis of PTT with Cutinase

-   -   Equipment: Launder-Ometer    -   Treatment pH: pH 8.6 (50 mM Tris Buffer)    -   Treatment temperature: 40° C.    -   Treatment time: 24 hours    -   Enzyme: Cutinase (Pseudomonas mendocina) @ 40 ppm    -   Control: Inactivated cutinase (Pseudomonas mendocina) @ 40 ppm    -   Substrates: scoured 100% Corterra®) brand PTT polyester

After the cutinase treatments, ⅝ inch disks were cut from the treatedswatches. The disks were incubated with 5 ppm subtilisin and 0.1%non-ionic surfactant (Triton X-100) to remove proteins bound ontopolyester. The increase in dyeability is shown in FIG. 9. From thisdata, it is apparent that the polyesterase modified the surfaceproperties of the PTT based fabric.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

1. A method for modifying a polyester comprising treating said polyesterwith a polyesterase enzyme for a time and under conditions to modify theproperties of said polyester.
 2. The method according to claim 1,wherein said polyester is a resin, film, fiber, yarn or fabric.
 3. Themethod according to claim 1, wherein said polyester is an aromaticpolyester.
 4. The method according to claim 2, wherein said polyesterfiber, yarn or fabric is a textile product and does not comprise astain.
 5. The method according to claim 1, wherein said polyesterase hasat least 10% greater hydrolysis in a UV and/or a MB assay than thecontrol.
 6. The method according to claim 5, wherein said polyesterasehas at least 50% greater hydrolysis in a UV and/or a MB assay than thecontrol.
 7. The method according to claim 6, wherein said polyesterasehas at least 100% greater hydrolysis in a UV and/or a MB assay than thecontrol.
 8. The method according to claim 4, wherein said textileproduct is modified in its properties of pilling, pilling prevention,weight, feel, appearance and/or luster.
 9. The method according to claim8, wherein said polyester textile is treated prior to the application ofa finish.
 10. The method according to claim 1, wherein said polyesteraseis derived from animal, plant, fungal or bacterial origin.
 11. Themethod according to claim 7, wherein said polyesterase is derived fromAbsidia spp.; Acremonium spp.; Agaricus spp.; Anaeromyces spp.;Aspergillus spp.; Aeurobasidium spp.; Cephalosporum spp.; Chaetomiumspp.; Coprinus spp.; Dactyllum spp.; Fusarium spp.; Gliocladium spp.;Helminthosporum spp.; Humicola spp.; Mucor spp.; Neurospora spp.;Neocallimastix spp.; Orpinomyces spp.; Penicillium spp; Phanerochaetespp.; Phlebia spp.; Piromyces spp.; Pseudomonas spp.; Rhizopus spp.;Schizophyllum spp.; Trametes spp.; Trichoderma spp.; and Ulocladiumspp.; Zygorhynchus spp.; Bacillus spp.; Cellulomonas spp.; Clostridiumspp.; Myceliophthora spp.; Pseudomonas spp.; Thermomonospora spp.;Thermomyces spp.; Streptomyces spp.; Fibrobacter spp.; Candida spp.;Pichia spp.; Rhodotorula spp.; or Sporobolomyces spp.
 12. A method forimproving the textile characteristics of a polyester article, comprisingthe steps of: (a) obtaining a polyesterase enzyme; (b) contacting saidpolyesterase enzyme with said polyester article under conditions and fora time suitable for said polyesterase to produce a modified polyesterarticle and produce a modified polyester article.
 13. The methodaccording to claim 9, wherein said polyester article comprises a fiber,yarn or fabric and said fiber yarn or fabric is subsequentlyincorporated into a textile.
 14. A polyester article produced accordingto the method of claim
 1. 15. The polyester article according to claim14, wherein said composition has an increased resistance to stains. 16.The polyester article according to claim 14, wherein subsequent to saidtreating, said composition is treated with a cationic compound.
 17. Theuse of polyesterase to improve the textile characteristics of apolyester.
 18. The method according to claim 1, wherein said treatmentoccurs in the presence of polypropylene glycol or glycerol.
 19. A methodor determining the polyesterase activity of a biological materialcomprising the steps of: (a) preparing an aqueous solution of abiological material; and (b) subjecting said aqueous compositioncomprising said biological material to conditions and for a time whereinit is determined whether said biological material comprises polyesteraseactivity.
 20. A kit for carrying out an assay for polyesterase activitycomprising: (a) a sample of polyester; (b) instructions for preparing abiological material for assaying whether said biological materialcomprises polyesterase activity.